The Center for Disease Control estimates that, as of 2011, almost 48 million people in the USA experience some form of food-borne illness every year. Of these, 128,000 are hospitalized and 3000 die. Worldwide, Campylobacter species are the most common cause of bacterial gastroenteritis, with 400-500 million cases of diarrhea each year. Infants in developing countries are at even greater risk, as are travelers to those countries. Campylobacter—associated gastroenteritis is estimated to affect nearly 1 million people a year in the USA. In approximately 1 in 1000 cases, Campylobacter jejuni is closely linked to the subsequent development of Guillian-Barre Syndrome, an acute, auto-immune paralysis. C. jejuni infection has also been associated with reactive arthritis in both children and adults.
In addition to Campylobacter spp., other common causes of bacterial food-borne gastroenteritis are enteropathogenic Escherichia coli, and Salmonella spp.; norovirus is a significant viral cause of food-borne disease. Although many cases of “food poisoning” self-resolve without the need for medical treatment, infants and the elderly are at higher risk for dehydration and more serious complications.
When individuals with severe symptoms of gastroenteritis seek medical help, the clinician is faced with multiple possible causes that can present with similar clinical features (e.g., diarrhea, nausea, vomiting, fever, abdominal pain) but that require very different treatment. For viruses, use of antibiotics is ineffective and can contribute to development of antibiotic resistance in bacteria. Ineffective empirical treatment can release selective pressure on resident resistant bacteria and convert non-symptomatic infection into illness. Treatment of enteropathogenic E. coli with antibiotics is contraindicated, as this may actually increase the chances of the patient experiencing hemolytic uremic syndrome (HUS). Antibiotic treatment of Salmonella-caused gastroenteritis may prolong the carrier state and is associated with relapse. Conversely, erythromycin treatment of Campylobacter infection may shorten illness and the time during which bacteria are shed, but only if the antibiotic is begun within 4 days of the onset of symptoms. Post-infectious irritable bowel syndrome has been associated with a longer duration of untreated infection. Identifying, without delay, which organism may be causing the gastroenteritis is important for diagnosis and selection of the appropriate treatment.
For Campylobacter, the current standard for identification is bacterial culture followed by microscopic examination of the organisms. Although this traditional method is straightforward, it has three major limitations. First, pathogenic species of Campylobacter are microaerophilic or strictly anaerobic, so that exposure of culture or stool to environmental oxygen conditions leads to death or inactivation of the bacteria. Thus, during transport or storage of specimens the number of viable organisms can decrease, leading to potentially inaccurate culture results. Second, while the standard culture medium and microaerophilic incubation conditions have been improved for two common species, Campylobacter jejuni (Cj) and Campylobacter coli (Cc), other Campylobacter species such as C. lari, C. upsaliensis, C. ureolyticus and C. hyointestinalis have also been linked to human disease, but are more difficult to grow and require different culture media and anaerobic incubation that are not routinely available in clinical laboratories. As a result, illnesses caused by these more fastidious, but never-the-less pathogenic organisms, are likely to go unrecognized and unreported. Third, even with appropriate culture conditions, each of these species is slow-growing, requiring from 48-72 hours (e.g., Cj or Cc) up to 7 days before reaching a point where the culture can safely be reported out as negative. Prolonged culture lengths are particularly true in the food safety industry, in which rinses of poultry carcasses or samples of raw milk (the most commonly identified sources of Campylobacter illnesses) are sampled for surveillance of sanitation measures or during outbreaks. Such delays can leave the clinician in a quandary and the patient with non-specific, ineffective, or even inappropriate treatment.
To provide a more rapid alternative to growth of cultures and microscopy for the identification and diagnosis of Campylobacter and campylobacteriosis, the present invention utilizes an outer membrane protein (OMP 18) as a biomarker for use in immunoassays of bacterial cultures or human fecal samples. The fecal samples used in the present invention may be unpreserved or preserved.
OMP 18 is an 18 kilodalton protein found in the outer membrane of many Campylobacter species. The amino acid sequence of OMP 18 is partially homologous to a similar protein, PAL in E. coli. The E. coli PAL protein is a peptidoglycan-associated lipoprotein, and the Campylobacter OMP18 is predicted to share these properties.
Several properties of OMP18 make it a good candidate for a diagnostic marker. Outer membrane proteins are typically subject to significant variation even among strains of the same species. The flagellin protein (FlaA), major outer membrane protein (MOMP/PorA) and fibronectin-binding protein (CadF) show large variability even between Campylobacter jejuni strains. However, from the genomic sequences of various strains of Cj that are currently available, the amino acids of OMP 18 (C jejuni subsp jejuni, ATTC 33560; GenBank: EIB41509.1) (FIG. 1) are completely identical among 51 strains of C. jejuni and 93% identical to the OMP18 protein of C. coli. OMP 18 thus provides a potential immunogen that is well-conserved among many strains of Cj and Cc. Conversely, OMP 18 bears little similarity to outer membrane proteins of Campylobacter species that are non-pathogenic or that are not clearly associated with human illness. Additionally, as a proposed antigen, OMP 18 has been shown to be highly immunogenic, a requirement for robust immunoassays. Serum from individuals that have had confirmed campylobacteriosis contains antibodies that recognize OMP 18 in immunoblots of bacterial cultures.
The present invention is directed towards improving ease of use and overcoming the limitations of delay inherent in Campylobacter bacterial culture. As previously stated, the current standard for identification of Campylobacter is through culturing and microscopy, which has several limitations, such as decreased viability with exposure to aerobic conditions. In contrast, in an embodiment of the present invention, a new method of detecting Campylobacter in stool samples using antibodies to OMP18 in immunoassays is provided. Antibody-based tests, such as Enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests, are rapid and cost-effective tests for detection of pathogen-specific antigens. Furthermore, this new method allows for the identification of Campylobacter in aerobic conditions, without culturing. Thus, the identification of Campylobacter can take place even when the sample is not alive. This is beneficial, for example, if a sample is brought into the lab under prolonged aerobic conditions. We propose to use OMP18 as an antigen, and antibodies developed using OMP18, for the detection of Campylobacter in stool.